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We report on the Xenopus homolog of the Hox 2.4 gene. This gene occupies the next to 5'-most position in the Xenopus Hox2 complex. Hox 2.4 RNA is first detected at the early neurula stage, reaching a peak at the early tailbud stage, and is localized in the middle and posterior portions of the embryos. Antibodies raised against a fusion protein show expression of Hox 2.4 protein in Xenopus embryos in a band located in the mid spinal cord. Thus, the protein is expressed in a narrower domain than that of Hox 2.4 mRNA. The Xenopus Hox 2.4 antibody cross-reacts readily with mouse embryonic tissue, where the protein is detected in migrating neural crest cells, the dorsal portion of the spinal cord, somites, lateral plate mesoderm, and in the forelimb bud. The Xenopus Hox 2.4 intron shares considerable sequence identity with the intron in the mouse homolog. A reporter gene containing an element from this intron which can bind homeodomain proteins is activated following microinjection into Xenopus embryos. The short distance between the end of the Hox 2.4 cDNA and the start site of the neighboring gene in the complex raises the possibility that this transcriptional element might be shared by two Hox genes.
Fig. 3. Temporal expression of the Hox 2.4 transcript in Xenopus
embryos. RNA was isolated from embryos of the indicated stages and
hybridized with antisense pS400 probe. The stages of the embryos are as
follows: lane 1, unfertilized egg; lane 2, gastrula (stage 11); lane 3,
neurula (stage 14); lane 4, early tailbud (stage 22); lane 5, late tailbud
(stage 28); lane 6, early tadpole (stage 32), according to Nieuwkoop and
Faber (1967). The amount of RNA loaded was monitored with the probe
for EF-la (elongation factor-la gene, Krieg et al., 1989).
Fig. 4. Regional expression of the Hox 2.4 transcript in Xenopus embryos.
The top part of the figure shows the dissected regions of stage 14
and 26 embryos used in the analysis of Hox 2.4 transcripts along the
anteroposterior axis of neurula and tailbud stage embryos. The bottom
part shows results of RNAse protection assays on the RNAs from the
dissected embryonic parts. Numbering for the lanes corresponds to the
number on the diagram labeling the segments dissected. Twenty embryos
of the indicated stages were manually dissected. Each lane contains
RNA pooled from five fragments. Note that Hox 2.4 transcripts are
localized in the middle to posterior region of the embryos. An actin probe
was used to normalize the RNA recovery.
Fig. 5. Tissue-specific expression of the Hox 2.4 transcript in normal
Xenopus embryos. The right panel depicts the regions of stage 24 embryos
dissected and subjected to RNAse protection analysis. Ventral
mesectoderm consists mostly of lateral plate mesoderm. The left panel
shows results of RNAse protection assays of the RNAs from the dissected
embryonic parts using pS400 as a probe. Each track contained
RNA from approximately five embryo fragments.
Fig. 6. Hox2.4 expression requires mesoderm induction. The top part
of the panel shows a diagram depicting the conjugate-forming procedure.
The bottom section shows the results of RNAse protection assays performed
on RNA from the fragments. A, animal fragment alone; V, vegetal
fragment alone; AN, animal/vegetal conjugate; N, RNA from normal neurulae
incubated for the same time as the conjugate. Cytoskeletal actin
was used to normalize the amount of RNA loaded; cardiac actin was used
to demonstrate the induction of mesoderm in only the whole neurulae and
AN conjugates. A, V, AN, and N lanes contain RNA from the equivalent
of five, two, two, and two embryos, respectively.
Fig. 7. Hox2.4 is induced following treatment with retinoic acid. Blastula
or gastrula stage embryos were treated with retinoic acid for 30
minutes. RNA was harvested at the neurula stage. Levels of neural induction
were measured using an N-CAM probe. The full-length Hox 2.4
protection fragment is indicated. A lower level of induction was observed
in embryos treated at the blastula as opposed to gastrula stage. The
experiment shown in the figure was done using M RA. However, a
similar result was seen with M RA. Each lane contained RNA from
two embryos. The asterisks indicate degradation products from the Hox
2.4 ('1) and N-CAM ('2) probes.
Fig. 8. Xenopus embryo wholemount antibody staining. Stage 26 tailbud
embryos were wholemount stained with Hox2.4 antibody. The region
of expression in the middle spinal cord is indicated with a bracket. Staining
of the notochord (No) occurs on the surface and is artifactual.
